Compositions and methods for detection of epstein barr virus (EBV)

a technology of epstein barr virus and composition method, applied in the field of in vitro diagnostics, can solve the problems of complex actual diversity characterization of the virus, particular risk to immunosuppressed transplant patients, and a threat to immunosuppressed patients

Pending Publication Date: 2022-08-11
ROCHE SEQUENCING SOLUTIONS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunosuppressed transplant patients are at particular risk as serious complications can arise from uncontrolled growth of latently EBV-infected B-cells leading to post-transplant lymphoproliferative disorder (PTLD).
EBV is one of several common (90%), often asymptomatic viral infections, but which p

Method used

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  • Compositions and methods for detection of epstein barr virus (EBV)
  • Compositions and methods for detection of epstein barr virus (EBV)
  • Compositions and methods for detection of epstein barr virus (EBV)

Examples

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example 1

Design of Primers and Probes for Detection of EBV by Real-Time PCR

[0080]The EBV nucleic acid test was designed with two targets in mind. The two targets chosen were BMRF2 and EBNA1, and are shown in FIG. 1. These candidates could be used individually or together duplexed in a dual target assay. If used as a dual target assay, two sets of primers and probe are employed (each set detecting either EBNA1 or BMRF2). As shown in Table 1, above, the primers for BMRF2 target have the nucleic acid sequence SEQ ID NOs:1 and 3, and the probe for BMRF2 target has the nucleic acid sequence of SEQ ID NO:2. Also, as shown in Table 1, above, the primers for EBNA1 target have the nucleic acid sequence SEQ ID NOs:4 and 6, and the probe for EBNA1 target has the nucleic acid sequence of SEQ ID NO:5. The amplicon generated by the primers targeting BMRF2 is a 96 base pair long amplicon, and is shown in FIG. 2 (along with locations where the primers and probes overlap the amplicon). The amplicon generated...

example 2

EBV Primers and Probes Detect BMRF2 and EBNA-1 of EBV in Real-Time PCR Assay

[0082]The EBV nucleic acid test was tested using primers / probes for detecting BMRF2 (SEQ ID NOs:1-3) and EBNA1 (SEQ ID NOs:4-6). A full process of the EBV assay was run. Four types of EBV-containing samples were employed: (1) EBV-infected Raji cells extracts spiked into EBV-negative plasma; (2) extracted EBV DNA (extracted from a B95-8 cell line from Advanced Biotechnologies (Catalog No. 17-926-500)); (3) Qnostics EBV Analytic Panel (EBV1604009C); and (4) the 1st WHO International Standard for EBV. Reagents used include cobas® 6800 / 8800 generic PCR Master Mix, with the profile and conditions for use with the cobas® 6800 / 8800, and using TaqMan® amplification and detection technology. The final concentration of oligonucleotides in the master mix was 0.3 μM for primers and 0.1 μM for probes. The cobas® 6800 / 8800 PCR Profile employed is depicted in Table 2, below:

TABLE 2cobas ® 6800 / 8800 PCR ProfileTargetHold ti...

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Abstract

Methods for the rapid detection of the presence or absence of Epstein Barr Virus (EBV) in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting EBV, and kits are provided that are designed for the detection of target regions of EBV. Also described are kits, reaction mixtures, and oligonucleotides (e.g., primer and probe) for the amplification and detection of EBV. Also described are primers and probes that detect different regions of EBV, and can be employed in a dual target assay for simultaneously detecting two different and non-overlapping target regions of EBV.

Description

FIELD OF THE INVENTION[0001]The present disclosure relates to the field of in vitro diagnostics. Within this field, the present invention concerns the amplification and detection of a target nucleic acid that may be present in a sample and particularly, the amplification, detection, and / or quantitation of a target nucleic acid comprising sequence variations and / or individual mutations of Epstein Barr Virus (EBV), using primers and probes. The invention further provides reaction mixtures and kits containing primers and probes for amplification and detection of EBV.BACKGROUND OF THE INVENTION[0002]Epstein Barr Virus (EBV) is a DNA virus, a member of the herpesvirus group (it has an alternate designation of Human Herpesvirus-4 or HHV-4). EBV infection is common, with an estimated 90% prevalence in adults, and is often asymptomatic. EBV infection can cause mononucleosis in its initial lytic stage, before entering one or more of several latent stages. EBV is highly linked with life threa...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6813C12Q1/6844
CPCC12Q1/70C12Q1/6844C12Q1/6813C12Q1/705C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/113C12Q1/701C12Q2531/113C12Q2561/101
Inventor HAMILTON, AARON T.HEIL, MARINTHALIGGETT, DEBRASUN, JINGTAOWANG, LINGWU, XIAONING
Owner ROCHE SEQUENCING SOLUTIONS INC
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